探討以蛇床子作為Cap對於羥基醯胺類化合物抑制組蛋白去乙醯酶之影響

Investigation of the effect of Osthole Capped to Hydroxamates on Histone Deacetylase Inhibitory Activity

吳暐凡(Wei-Fan Wu)

指導教授 ： 黃偉展

臺北醫學大學/藥學院/生藥學研究所/碩士(2012年)

https://doi.org/10.6831/TMU.2012.00160

摘要

組織蛋白乙醯化修飾係藉由組織蛋白乙醯基轉移酶 (HATs)及組織蛋白去乙醯酶 (HDACs)這兩類酵素活性來達到調控平衡。組織蛋白去乙醯酶表現異常為許多癌症之病理原因。實驗發現組織蛋白去乙醯酶抑制劑對不同癌細胞株及動物體內試驗中，均顯現抗癌活性，被證實為一種癌症治療策略。
目前所發表之組織蛋白去乙醯酶抑制劑之主結構可分為三部分：(1)供特定類別組織蛋白去乙醯酶表面結合之疏水性辨識端、(2)與活性位通道內底部鋅離子螯合之基團、(3)介於兩者間，長度約四至六個碳之疏水性連結碳鏈。過去我們曾經發表以C3-C4還原後之蛇床子素衍生物為Cap加入肉桂羥基醯胺骨架中，發現能明顯提升組織蛋白去乙醯酶抑制活性。因此我們嘗試將其C3-C4未氧化導入目前正進行臨床試驗之組織蛋白去乙醯酶抑制劑長鏈羥基醯胺及肉桂酸羥基醯胺，合成一系列新的組織蛋白去乙醯酶抑制劑。
將合成之化合物進行前列腺癌PC-3細胞生長抑制實驗及測試它們對於數種組織蛋白去乙醯酶異構體之抑制活性。結果顯示取代基為長鏈羥基醯胺類化合物13a與13b，對於海拉細胞核萃取物 (HeLa nuclear extract) 之組織蛋白去乙醯酶抑制活性約為肉桂酸羥基醯胺取代者8a與8b之兩倍，而8a與8b之抑制活性則與正對照品SAHA相當。對位取代8b與13b抑制組織蛋白去乙醯異構酶4活性之效果則明顯優於間位取代8a與13a及對照品SAHA。此外，長鏈羥基醯胺與肉桂酸羥基醯胺兩類蛇床子素衍生之羥基醯胺類化合物間就組織蛋白去乙醯異構酶8之酵素抑制活性及人類前列腺癌細胞PC-3之生長抑制活性無顯著差異。
分子模擬嵌合運算結果顯示，化合物8a, 8b, 13a與13b和組織蛋白去乙醯異構酶4結合後其能量相近 (-7.8~-7.6 Kcal/mol)；而組織蛋白去乙醯異構酶8對接結果顯示肉桂酸羥基醯胺類比長鏈羥基醯胺類與目標蛋白有較佳之結合親和力 (-7.8~-7.4 Kcal/mol<-7.1~-7.0 Kcal/mol)。以先前實驗室發表過之四個蛇床子素衍生物6c, 6d, 6g與6k對組織蛋白去乙醯酶類似蛋白進行分子模擬嵌合，整體結合親和力最差者為6k (-5.8 Kcal/mol)，其餘三者同為-6.5 Kcal/mol。在具組織蛋白去乙醯酶抑制活性之天然物虛擬篩選中，剃除無適當嵌合模式者後，仍以含羥基醯胺基團之Psammplin A (-11.2 Kcal/mol) 親和力鍵結能量最佳，其次為黃酮類之Aurantinidin (-9.8 Kcal/mol) 與Pomiferin (-9.2 Kcal/mol) ，最差者為非六員環之Depudecin (-6.2 Kcal/mol)。

關鍵字

蛇床子 ； 羥基醯胺 ； 組蛋白去乙醯酶

並列摘要

Histone acetylation is balanced by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The abnormal expressions of HDACs may be implicated with many cancers. Studies revealed that HDAC inhibitors showed anticancer effect on cancer cells as well as animal models, thus, it was considered as strategy of cancer treatment.
The structure of recently reported HDAC inhibitors can be divided into three motifs: (1) cap, which was recognized for specific subtype HDAC surface at the rim of active-site cavity (2) zinc binding group (ZBG) , which chelates Zn2+ in the bottom of active-site cavity (3) linker, which connects Cap to ZBG. The optimal length of linker is approximated 4-6 carbon. Previously, our lab have reported a series of N-hydroxycinnamides with C3-C4 saturated osthole derivative as a Cap. We found that some showed significant inhibitory activity against HDAC. In the present study, we planned to extend to C3-C4 unsaturated osthole derivative incorporated into N-hydroxycinnamide and suberoylanilide hydroxamic acid, two common scaffolds used for clinical HDAC inhibitors.
The synthesized compounds were evaluated for anticancer activities against human prostate cancer PC-3 cells and HDAC inhibition. The enzyme inhibitory result showed that suberoylanilide hydroxamic acids 13a and 13b were about two-fold more potent than N-hydroxycinnamides 8a, 8b in the test against HeLa cell nuclear HDAC using SAHA as a positive control. With respect to HDAC4, para-substituted compounds 8b and 13b exhibited higher potency than meta-substituted 8a, 13a and SAHA. It was found that no significant difference was observed between two scaffolds in HDAC 8 inhibition and growth inhibitory activity against PC-3 cells.
Molecular modeling revealed that compounds 8a, 8b, 13a, and 13b had similar binding energy (-7.8~-7.6 Kcal/mol) in docking to HDAC 4; regarding to HDAC 8, N-hydroxycinnamides 8a-b showed higher binding affinity than suberoylanilide hydroxamic acids 13a-b. We chose four osthole-based compounds, 6c, 6d, 6g, and 6k, previously reported by our lab, for docking to histone deacetylase like protein (HDLP); the weakest binding energy was for 6k (-5.8 Kcal/mol), others were all -6.5 Kcal/mol. In the virtual screening for natural products with HDAC inhibitory activity, Psammplin A (-11.2 Kcal/mol) with hydroxamate structure showed best binding affinity, the next two were flavonoids, Aurantinidin (-9.8 Kcal/mol), and Pomiferin (-9.2 Kcal/mol), and Depudecin (-6.2 Kcal/mol) was the last one.

並列關鍵字

osthole ； hydroxamates ； histone deacetylase

參考文獻

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